Compositions and methods for helper-free production of recombinant adeno-associated viruses

ABSTRACT

A method for producing recombinant adeno-associated virus in the absence of contaminating helper virus or wild-type virus involves culturing a mammalian host cell containing an rAd/AAV hybrid virus, an AAV rep sequence and an AAV cap sequence under the control of regulatory sequences directing expression thereof. The rAd/AAV hybrid virus contains a rAAV construct to be packaged into an AAV virion in a backbone containing the adenoviral sequences necessary to express E1a and E1b gene products and to permit replication of the hybrid virus. The method of the invention permits replication of the hybrid virus and production of rAAV virion in this host cell in the absence of a helper virus and obviates a subsequent purification step to purify rAAV from contaminating virus.

CROSS-REFERENCE TO RELATED APPLICATIONS

This is a continuation-in-part of International Patent ApplicationPCT/US99/05870, filed Mar. 18, 1999.

BACKGROUND OF THE INVENTION

Adeno-associated virus (AAV) is a replication-deficient parvovirus, thegenome of which is about 4.6 kb in length, including 145 nucleotideinverted terminal repeats (ITRs). Two open reading frames encode aseries of rep and cap polypeptides. Rep polypeptides (rep78, rep68,rep62 and rep40) are involved in replication, rescue and integration ofthe AAV genome. The cap proteins (VP1, VP2 and VP3) form the virioncapsid. Flanking the rep and cap open reading frames at the 5′ and 3′ends are 145 bp inverted terminal repeats (ITRs), the first 125 bp ofwhich are capable of forming Y- or T-shaped duplex structures. Ofimportance for the development of AAV vectors, the entire rep and capdomains can be excised and replaced with a therapeutic or reportertransgene [B. J. Carter, in “Handbook of Parvoviruses”, ed., P. Tijsser,CRC Press, pp.155-168 (1990)]. It has been shown that the ITRs representthe minimal sequence required for replication, rescue, packaging, andintegration of the AAV genome.

When this nonpathogenic human virus infects a human cell, the viralgenome integrates into chromosome 19 resulting in latent infection ofthe cell. Production of infectious virus and replication of the virusdoes not occur unless the cell is coinfected with a lytic helper virus,such as adenovirus (Ad) or herpesvirus. Upon infection with a helpervirus, the AAV provirus is rescued and amplified, and both AAV andhelper virus are produced. The infecting parental ssDNA is expanded toduplex replicating form (RF) DNAs in a rep dependent manner. The rescuedAAV genomes are packaged into preformed protein capsids (icosahedralsymmetry approximately 20 nm in diameter) and released as infectiousvirions that have packaged either + or − ss DNA genomes following celllysis.

AAV possesses unique features that make it attractive as a vector fordelivering foreign DNA (i.e., a transgene) to cells, and various groupshave studied the potential use of AAV in the treatment of diseasestates. As used in this application, the term “transgene” means the DNAdesired to be delivered to an animal, the DNA being non-AAV DNA.However, progress towards establishing AAV as a transducing vector forthe delivery of DNA in the form of a desired transgene has been slow fora variety of reasons.

One obstacle to the use of AAV for delivery of DNA has been lack ofhighly efficient schemes for encapsidation of recombinant genomes andproduction of infectious virions. See, R. Kotin, Hum. Gene Ther.,5:793-801 (1994)]. One method which addresses this problem involvestransfecting a recombinant AAV (rAAV) (which has the DNA to bedelivered, but lacks rep and cap genes) into host cells followed byco-infection with wild-type (wt) AAV (which supplies the rep and capgenes) and adenovirus (which supplies at least the four adenovirusgenes: E1, E2, E4 and VAI, which have been stated to be necessary forrAAV production) [see, e.g., Carter, cited above]. However, this methodrequires mandatory co-infection and leads to unacceptably high levels ofwt AAV resulting from non-homologous recombination and contamination ofthe rAAV produced with wt AAV. The contamination with other viruses orplasmids demands purification of rAAV. Incubation of cells with rAAV inthe absence of contaminating wt AAV or helper adenovirus yields littlerecombinant gene expression.

A widely recognized means for manufacturing transducing AAV virions forgene therapy entails co-transfection with two different, complementingplasmids. One of these plasmids contains a therapeutic or reportertransgene sandwiched between the two cis acting AAV ITRs. The AAVcomponents that are needed for rescue and subsequent packaging ofprogeny recombinant genome are provided in trans by a second plasmidencoding the viral open reading frames for rep and cap proteins. In thissystem, the Ad helper functions are provided by a wt adenovirus or byreplication-defective adenovirus with the missing E1 gene supplied byHEK 293 cells. Other variants of this method have been described. See,for example, U.S. Pat. No. 5,658,785, which refers to a mammalian hostcell stably transfected with a rAAV genome and with AAV rep and capgenes, and a method for producing rAAV by infecting this host cell witha helper virus.

U.S. Pat. No. 5,658,776 refers to packaging systems and processes forpackaging AAV vectors in which the AAV p5 promoter is replaced with aheterologous promoter. Alternatively, U.S. Pat. No. 5,622,856 refers toconstructs and methods for AAV vector production in which the homologousp5 promoter is moved to a position 3′ of the rep genes, optionallyflanking the rep/cap genes and repositioned p5 promoter with FRTsequences.

There remains a need in the art for additional compositions and methodspermitting the efficient production of AAV and recombinant AAV virusesfor use as vectors for somatic gene therapy without the inefficiency,contamination and purification problems present in the methodspreviously described.

SUMMARY OF THE INVENTION

The present invention allows for the efficient production of rAAVcontaining a desired transgene DNA. Particularly, the present inventionprovides both compositions and methods which enable the production of arAAV without the problem of homologous recombination which producescontaminating re-assembled wt AAV during rAAV production.

In one aspect, the invention provides a replication-competent hybridadenovirus/AAV virus containing a recombinant adeno-associated viral(rAAV) vector and sufficient adenoviral sequences to permit replicationof said hybrid virus in a selected host cell. In one embodiment, thehybrid virus contains a functional deletion in the wild-type adenoviralE3 region and/or a non-functional deletion of adenoviral sequences inthe E4 region, such that the hybrid virus contains an adenoviral E4 ORF6region.

In another aspect, the invention provides an adenovirus/AAV hybrid viruscontaining (a) adenovirus 5′ cis-elements necessary for replication andpackaging; (b) a recombinant adeno-associated viral (rAAV) vector; (c) adeletion of adenoviral sequences from the E3 region; (d) nucleic acidsequences encoding adenovirus E1a and adenovirus E1b under the controlof regulatory sequences directing expression of the E1a and E1b geneproducts, wherein said E1a and E1b nucleic acid sequences are located inthe site of the E3 region and; (e) adenovirus 3′ cis-elements necessaryfor replication and packaging. In one suitable embodiment, the hybridvirus provides all sequences necessary to provide helper function forpackaging the rAAV.

In another aspect, a method for producing recombinant adeno-associatedvirus (rAAV) in the absence of contaminating helper virus or wild-typevirus, comprising the step of culturing a host cell comprising areplication-competent rAd/AAV and an AAV rep sequence and an AAV capsequence under the control of regulatory sequences directing expressionthereof. Suitably, the method further involves the step of controllingreplication of the rAd/AAV hybrid following infection, thereby enhancingproduction of rAAV. In a preferred embodiment, the method of theinvention simplifies purification steps because thereplication-competent rAd/AAV provides the rAAV construct to be packagedand all necessary adenoviral sequences, there is no helper virus usedand there is insufficient adenovirus sequence in the host cell to permithomologous recombination to a contaminating wt virus.

Other aspects and advantages of the present invention are describedfurther in the following detailed description of the preferredembodiments thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1B are a schematic map illustrating production of areplication-competent rAd/AAV hybrid virus.

BRIEF DESCRIPTION OF THE INVENTION

1. E1-Expressing Adenovirus/AAV Hybrid Virus

The present invention provides a recombinant adenovirus/AAV (rAd/AAV)hybrid virus, in which an adenovirus is engineered to contain a rAAVconstruct to be packaged into a rAAV virion and sufficient adenoviralsequences to permit replication of said hybrid virus in a selected hostcell. In a preferred embodiment, all the necessary adenoviral genes areprovided by the rAd/AAV hybrid virus, which may lack the adenoviralsequences from the wild-type E3 and may contain non-functional deletionsof adenoviral sequences, e.g., the hybrid virus may lack all adenoviralE4 coding sequences with the exception of the sequences necessary toexpress the E4 ORF6 function. The rAAV construct may be inserted intoeither the adenovirus E3 or the E1 region. Optionally, the sequencesencoding the E1 gene products are inserted into and expressed from thewild-type E3 region. In another embodiment, the invention provides arAd/AAV expressing the E1 gene products, but which lack one or more ofthe other necessary adenoviral sequences (e.g., E2a and/or E4 ORF6),which is supplied by the host cell The hybrid virus of the invention,described in detail below, is useful for production of rAAV in a hostcell which contains, at a minimum, an AAV rep sequence and an AAV capsequence under the control of regulatory sequences directing expressionthereof.

A. Adenoviral Gene Sequences

At a minimum, the adenovirus sequences employed in the rAd/AAV hybridvirus of the invention are the cis-acting 5′ and 3′ inverted terminalrepeat (ITR) sequences of an adenovirus (which function as origins ofreplication), the native 5′ packaging/enhancer domain, that containssequences necessary for packaging linear Ad genomes and enhancerelements for the E1 promoter, and the DNA necessary to permitreplication of the hybrid virus in a selected host cell. Mostpreferably, all necessary adenovirus gene products, i.e., E1a, E1b, E2a,E2b, E4 ORF6, the adenoviral intermediate genes IX and IXa, andadenoviral late genes L1, L2, L3, L4 and L5 are expressed from thehybrid virus. The adenovirus sequences may be derived from one or morewild-type adenoviruses or a mutant adenoviruses.

The DNA sequences encoding the adenoviral genes useful in this inventionmay be selected from among any known adenovirus type, including thepresently identified 46 human types [see, e.g., Horwitz, cited above andAmerican Type Culture Collection]. Similarly, adenoviruses known toinfect other animals may supply the gene sequences. The selection of theadenovirus type for each E1 and E2a gene sequence does not limit thisinvention. The sequences for a number of adenovirus serotypes, includingthat of serotype Ad5, are available from Genbank. A variety ofadenovirus strains are available from the American Type CultureCollection (ATCC), Manassas, Va., or are available by request from avariety of commercial and institutional sources. Any one or more ofhuman adenoviruses Types 1 to 46 may supply any of the adenoviralsequences. In the following exemplary embodiment the E1 and E2a genesequences are those from adenovirus serotype 5 (Ad5).

As used herein by “DNA which expresses the gene product”, it is meantany nucleic acid sequence the gene product or any functional portionthereof. Similarly included are any alleles or other modifications ofthe nucleic acid sequence (e.g., a gene) or functional portion thereof.As defined herein, “functional portion or functional fragment” is thatregion of coding sequence or gene product which is required to providethe necessary desired function. Such modifications may be deliberatelyintroduced by resort to conventional genetic engineering or mutagenictechniques to enhance the function of the gene product(s) in somemanner, as well as naturally occurring allelic variants thereof.

In a preferred embodiment, the rAd/AAV hybrid virus of the inventionlacks the sequences encoding the adenoviral E3 gene product, thusenabling a heterologous sequence to be inserted into the native E3region, which is non-essential for replication and infection. Such aheterologous sequence may be any nucleic acid sequence which is notnative to the adenovirus, or any nucleic acid sequence from anon-contiguous region of the same adenovirus. Similarly, the rAd/AAVhybrid virus may lack adenoviral E4 coding sequences with the exceptionof E4 ORF6 which is essential. Suitably, the rAd/AAV hybrid virus isengineered such that it does not exceed 105% of the size of the nativeadenoviral genome (e.g., 105% of 36 kb). Thus, where desired, e.g., topermit insertion of a desired heterologous sequence, the rAd/AAV may beengineered to contain other non-functional deletions of sequenceswild-type to the adenovirus(es) from which the hybrid is engineered.Such non-functional deletions include those which do not extinguish theability of the hybrid virus to express a functional gene productrequired for replication of the hybrid virus. The deletion of alladenoviral E4 coding sequences with the exception of the E4 ORF6sequences is an example of a non-functional deletion. Other suitablenon-functional deletions may be readily designed.

Thus, in particularly desirable embodiment, all the necessary adenoviralgenes for replication of the rAd/AAV hybrid virus are provided by thehybrid virus itself. Such a hybrid virus contains a rAAV construct to bepackaged into a virion, lacks adenoviral sequences encoding adenovirusE3, and lacks all adenoviral E4 coding sequences with the exception ofE4 ORF6 function. Optionally, the adenoviral E1a and E1b gene productsmay be expressed from a region other than the wild-type E1 region, e.g.,these gene products are inserted into and expressed from the wild-typeE3 region.

In another embodiment, the rAd/AAV of the invention lacks one or more ofthe other adenoviral genes required for replication. Any requiredadenoviral gene products which have been deleted from the hybrid viruscan be supplied in the production process by a selected packaging cellor in trans by a nucleic acid sequence directing expression of thedesired gene product. Preferably, the rAd/AAV expresses the E1a and E1bgene products. In one example, the rAd/AAV hybrid virus containswild-type E1 functions, but lacks the sequences necessary for expressionof E4 ORF6. In this situation, the rAd/AAV hybrid is introduced into ahost cell which contains the sequences necessary for expressing E4 ORF6.In another example the rAd/AAV hybrid virus lacks the sequencesnecessary for expression of E2a, which function is expressed in the hostcell used for production of the rAAV. Such host cells are discussed inmore detail below.

Design of these and other rAd/AAV hybrid viruses within the scope ofthis invention includes appropriate sequences that are operably linkedto the gene of interest to promote its expression. “Operably linked”sequences include both expression control sequences that are contiguouswith the gene of interest and expression control sequences that act intrans or at a distance to control the gene of interest. Such expressioncontrol sequences are discussed in more detail in connection with thetransgene.

The promoters for each of the adenoviral genes may be selectedindependently from a constitutive promoter, an inducible promoter or anative adenoviral promoter (i.e., a promoter may be one which isnaturally associated with the 5′ flanking region of an adenovirus gene).The promoters may be regulated by a specific physiological state of theorganism or cell (i.e., by the differentiation state or in replicatingor quiescent cells) or by exogenously-added factors, for example. Theselected promoters may be identical or may be different.

In one embodiment, the E1a gene (and subsequently the E1b gene) isexpressed under the control of a constitutive promoter, including,without limitation, the RSV LTR promoter/enhancer, the CMV immediateearly promoter/enhancer, the SV40 promoter, the dihydrofolate reductasepromoter, the cytoplasmic β-actin promoter and the phosphoglycerolkinase (PGK) promoter.

In another embodiment, an inducible promoter is employed to express theE1 gene products, so as to control the amount and timing of the cell'sproduction of the E1a and E1b gene products, which can be toxic to thecell upon excessive accumulation [see, e.g., William S. M. Wold, J. CellBiochem., 53:329-335 (1993); J. Nevins, Current Opinion in Genetics andDevelopment, 4:130-134 (1994); E. Harrington et al, Current Opinion inGenetics and Development, 4:120-129 (1994); G. Evan et al, CurrentOpinion in Cell Biology, 7:825-834 (1995); J. Nevins, Science, 258:424(1992)]. Inducible promoters include those known in the art and thosediscussed above including, without limitation, the zinc-inducible sheepmetallothionine (MT) promoter; the dexamethasone (Dex)-inducible mousemammary tumor virus (MMTV) promoter; the T7 promoter; the ecdysoneinsect promoter; the tetracycline-repressible system; thetetracycline-inducible system; the RU486-inducible system; and therapamycin-inducible system. Any type of inducible promoter which istightly regulated and which provides for high-level expression of E1 maybe used. Other types of inducible promoters which may be useful in thiscontext are those which are regulated by a specific physiological state,e.g., temperature, acute phase, a particularly differentiation state ofthe cell, or in replicating cells only.

B. Recombinant AAV Construct

In the rAd/AAV virus, the AAV sequences are inserted into the adenoviralbackbone in a region deleted of native adenoviral sequence, and thus areflanked by the selected adenoviral sequences. The AAV sequences aretypically in the form of a rAAV construct (e.g., a cassette) which ispackaged into a rAAV virion according to the method of the invention.The rAAV construct contains, at a minimum (from 5′ to 3′), 5′ AAV ITRsequences, a selected transgene under the control of a selected promoterand other conventional vector regulatory components, and 3′ AAV ITRsequences. Each of these components of the rAAV construct is discussedbelow. See, also, U.S. Pat. Nos. 5,856,152 and 5,871,982. One of skillin the art can readily engineer the rAd/AAV hybrid virus so as to insertthe rAAV construct into the desired adenoviral region. In oneembodiment, the rAAV construct is located in the adenoviral E3 region ofthe rAd/AAV hybrid virus. In another suitable embodiment, the rAAVconstruct is located in the adenoviral E1 region of the rAd/AAV hybridvirus. However, the present invention is not limited to these exemplaryembodiments.

1. AAV Sequences

The AAV sequences employed are preferably the cis-acting 5′ and 3′inverted terminal repeat sequences [See, e.g., B. J. Carter, in“Handbook of Parvoviruses”, ed., P. Tijsser, CRC Press, pp.155-168(1990)]. The ITR sequences are about 145 bp in length. Preferably,substantially the entire sequences encoding the ITRs are used in themolecule, although some degree of minor modification of these sequencesis permissible. The ability to modify these ITR sequences is within theskill of the art. [See, e.g., texts such as Sambrook et al, “MolecularCloning. A Laboratory Manual”, 2d ed., Cold Spring Harbor Laboratory,New York (1989); Carter et al, cited above; and K. Fisher et al., J.Virol., 70:520-532 (1996)]. An example of such a molecule employed inthe present invention is a “cis-acting” plasmid containing thetransgene, in which the selected transgene sequence and associatedregulatory elements are flanked by the 5′ and 3′ AAV ITR sequences.

The AAV ITR sequences may be obtained from any known AAV, includingpresently identified human AAV types. Similarly, AAVs known to infectother animals may also provide these ITRs employed in the molecules orconstructs of this invention. For example, the ITRs may be provided byAAV type 1, AAV type 2, AAV type 3, AAV type 4, AAV type 5, AAV 6, otherAAV serotypes or densovirus. A variety of AAV strains are available fromthe American Type Culture Collection or are available by request from avariety of commercial and institutional sources. In the followingexemplary embodiments an AAV-2 is used for convenience. However, theselection of the species and serotype of AAV that provides thesesequences is within the skill of the artisan according to the teachingsof this application and does not limit the following invention.

2. Transgene

According to the present invention, the rAAV construct contains anucleic acid molecule which comprises a desired transgene, a promoter,and other regulatory elements which control and direct expression of thetransgene in a host cell, flanked by AAV sequences. The transgenesequence is a nucleic acid sequence, heterologous to the AAV sequence,which encodes a polypeptide or protein of interest. The composition ofthe transgene sequence depends upon the intended use for the resultingrAAV. For example, one type of transgene sequence comprises a reporteror marker sequence, which upon expression produces a detectable signal.Such reporter or marker sequences include, without limitation, DNAsequences encoding β-lactamase, β-galactosidase (LacZ), alkalinephosphatase, thymidine kinase, green fluorescent protein (GFP),chloramphenicol acetyltransferase (CAT), luciferase, membrane boundproteins including, for example, CD2, CD4, CD8, the influenzahemagglutinin protein, and others well known in the art, to which highaffinity antibodies directed to them exist or can be made routinely, andfusion proteins comprising a membrane bound protein appropriately fusedto an antigen tag domain from, among others, hemagglutinin or Myc.

These sequences, when associated with regulatory elements which drivetheir expression, provide signals detectable by conventional means,including enzymatic, radiographic, colorimetric, fluorescence or otherspectrographic assays, fluorescent activated cell sorting assay andimmunological assays, including ELISA, RIA and immunohistochemistry. Forexample, where the transgene is the LacZ gene, the presence of rAAV isdetected by assays for beta-galactosidase activity. Similarly, where thetransgene is luciferase, rAAV may be measured by light production in aluminometer.

However, desirably, the transgene is a non-marker gene which can bedelivered to a cell or an animal via the rAAV produced by this method.The transgene may be selected from a wide variety of gene productsuseful in biology and medicine, such as proteins, antisense nucleicacids (e.g., RNAs), or catalytic RNAs. The invention may be used tocorrect or ameliorate gene deficiencies, wherein normal genes areexpressed but at less than normal levels, and may also be used tocorrect or ameliorate genetic defects wherein a functional gene productis not expressed. A preferred type of transgene sequence is atherapeutic gene which expresses a desired gene product in a host cell.These therapeutic nucleic acid sequences typically encode productswhich, upon expression, are able to correct or complement an inheritedor non-inherited genetic defect, or treat an epigenetic disorder ordisease. However, the selected transgene may encode any productdesirable for study. The selection of the transgene sequence is not alimitation of this invention. Choice of a transgene sequence is withinthe skill of the artisan in accordance with the teachings of thisapplication.

The invention also includes methods of producing rAAV which can be usedto correct or ameliorate a gene defect caused by a multi-subunitprotein. In certain situations, a different transgene may be used toencode each subunit of the protein. This is desirable when the size ofthe DNA encoding the protein subunit is large, e.g., for animmunoglobulin or the platelet-derived growth factor receptor. In orderfor the cell to produce the multi-subunit protein, a cell would beinfected with rAAV containing each of the different subunits.Alternatively, different subunits of a protein may be encoded by thesame transgene. In this case, a single transgene would include the DNAencoding each of the subunits, with the DNA for each subunit separatedby an internal ribosome entry site (IRES). This is desirable when thesize of the DNA encoding each of the subunits is small, such that thetotal of the DNA encoding the subunits and the IRES is less than fivekilobases.

Useful gene products include hormones and growth and differentiationfactors including, without limitation, insulin, glucagon, growth hormone(GH), parathyroid hormone (PTH), growth hormone releasing factor (GRF),follicle stimulating hormone (FSH), luteinizing hormone (LH), humanchorionic gonadotropin (hCG), vascular endothelial growth factor (VEGF),angiopoietins, angiostatin, granulocyte colony stimulating factor(GCSF), erythropoietin (EPO), connective tissue growth factors (CTGF),basic fibroblast growth factor (bFGF), acidic fibroblast growth factor(aFGF), epidermal growth factor (EGF), transforming growth factor a(TGFα), platelet-derived growth factor (PDGF), insulin-like growthfactors I and II (IGF-I and IGF-II), any one of the transfomiing growthfactor β (TGFβ) superfamily comprising TGFβ, activins, inhibins, or anyof the bone morphogenic proteins (BMP) BMPs 1-15, any one of theheregulin/neuregulin/ARIA/neu differentiation factor (NDF) family ofgrowth factors, nerve growth factor (NGF), brain-derived neurotrophicfactor (BDNF), neurotrophins NT-3 and NT-4/5, ciliary neurotrophicfactor (CNTF), glial cell line derived neurotrophic factor (GDNF),neurturin, agrin, any one of the family of semaphorins/collapsins,netrin-1 and netrin-2, hepatocyte growth factor (HGF), ephrins, noggin,sonic hedgehog and tyrosine hydroxylase.

Other useful gene products include proteins that regulate the immunesystem including, without limitation, cytokines and lymphokines such asthrombopoietin (TPO), interleukins (IL) IL-1α, IL-1β, IL-2, IL-3, IL4,IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15,IL- 16, and IL-17, monocyte chemoattractant protein (MCP-1), leukemiainhibitory factor (LIF), granulocyte-macrophage colony stimulatingfactor (GM-CSF), Fas ligand, tumor necrosis factors α and β (TNFα andTNFβ), interferons (IFN) IFN-α, IFN-β and IFN-γ, stem cell factor,flk-2/flt3 ligand. Gene products produced by the immune system are alsoencompassed by this invention. These include, without limitations,immunoglobulins IgG, IgM, IgA, IgD and IgE, chimeric immunoglobulins,humanized antibodies, single chain antibodies, T cell receptors,chimeric T cell receptors, single chain T cell receptors, class I andclass II MHC molecules, as well as engineered MHC molecules includingsingle chain MHC molecules. Use gene products also include complementregulatory proteins such as complement regulatory proteins, membranecofactor protein (MCP), decay accelerating factor (DAF), CR1, CR2 andCD59.

Still other useful gene products include any one of the receptors forthe hormones, growth factors, cytokines, lymphokines, regulatoryproteins and immune system proteins. The invention encompasses receptorsfor cholesterol regulation, including the LDL receptor, HDL receptor,VLDL receptor, and the scavenger receptor. The invention alsoencompasses gene products such as steroid hormone receptor superfamilyincluding glucocorticoid receptors and estrogen receptors, Vitamin Dreceptors and other nuclear receptors. In addition, useful gene productsinclude transcription factors such as jun, fos, max, mad, serum responsefactor (SRF), AP-1, AP-2, myb, MRG1, CREM, Alx4, FREAC1, NF-κB, membersof the leucine zipper family, C2H4 zinc finger proteins, includingZif268, EGR1, EGR2, C6 zinc finger proteins, including theglucocorticoid and estrogen receptors, POU domain proteins, exemplifiedby Pit1, homeodomain proteins, including HOX-1, basic helix-loop-helixproteins, including myc, MyoD and myogenin, ETS-box containing proteins,TFE3, E2F, ATF1, ATF2, ATF3, ATF4, ZF5, NFAT, CREB, HNF4, C/EBP, SP1,CCAAT-box binding proteins, interferon regulation factor 1 (IRF-1),Wilms tumor protein, ETS-binding protein, STAT, GATA-box bindingproteins, e.g., GATA-3, and the forkhead family of winged helixproteins.

Other useful gene products include carbamoyl synthetase I, ornithinetranscarbamylase, arginosuccinate synthetase, arginosuccinate lyase,arginase, fumarylacetoacetate hydrolase, phenylalanine hydroxylase,alpha-1 antitrypsin, glucose-6-phosphatase, low-density-lipoproteinreceptor, porphobilinogen deaminase, factor VIII, factor IX, cystathionebeta-synthase, branched chain ketoacid decarboxylase, albuminisovaleryl-CoA dehydrogenase, propionyl CoA carboxylase, methyl malonylCoA mutase, glutaryl CoA dehydrogenase, insulin, beta-glucosidase,pyruvate carboxylase, hepatic phosphorylase, phosphorylase kinase,glycine decarboxylase (also referred to as P-protein), H-protein,T-protein, Menkes disease protein, tumor suppressors (e.g., p53), cysticfibrosis transmembrane regulator (CFTR), and the product of Wilson'sdisease gene PWD.

Other useful transgenes include non-naturally occurring polypeptides,such as chimeric or hybrid polypeptides or polypeptides having anon-naturally occurring amino acid sequence containing insertions,deletions or amino acid substitutions. For example, single-chainengineered immunoglobulins could be useful in certain immunocompromisedpatients. Other types of non-naturally occurring gene sequences includeantisense molecules and catalytic nucleic acids, such as ribozymes,which could be used to reduce overexpression of a gene.

Design of the transgene for expression in mammalian cells and hostsshould include appropriate sequences that are operably linked to thegene of interest to promote its expression. Expression control sequencesinclude appropriate transcription initiation, termination, promoter andenhancer sequences; efficient RNA processing signals such as splicingand polyadenylation signals; sequences that stabilize cytoplasmic mRNA;sequences that enhance translation efficiency (i.e., Kozak consensussequence); sequences that enhance protein stability; and when desired,sequences that enhance protein secretion. A great number of expressioncontrol sequences—native, constitutive, inducible and/ortissue-specific—are known in the art and may be utilized to driveexpression of the transgene, depending upon the type of expressiondesired. For eukaryotic cells, expression control sequences typicallyinclude a promoter, an enhancer, such as one derived from animmunoglobulin gene, SV40, cytomegalovirus, etc., and a polyadenylationsequence which may include splice donor and acceptor sites. Thepolyadenylation sequence generally is inserted following the transgenesequences and before the 3′ AAV ITR sequence. A rAAV construct useful inthe present invention may also contain an intron, desirably locatedbetween the promoter/enhancer sequence and the transgene. One possibleintron sequence is also derived from SV-40, and is referred to as theSV-40 T intron sequence. Another vector element that may be used is aninternal ribosome entry site (IRES). An IRES sequence is used to producemore than one polypeptide from a single gene transcript. An IRESsequence would be used to produce a protein that contain more than onepolypeptide chains. Selection of these and other common vector elementsare conventional and many such sequences are available [see, e.g.,Sambrook et al, and references cited therein at, for example, pages3.18-3.26 and 16.17-16.27 and Ausubel et al., Current Protocols inMolecular Biology, John Wiley & Sons, New York, 1989].

In one embodiment, high-level constitutive expression will be desired.Examples of such promoters include, without limitation, the retroviralRous sarcoma virus (RSV) LTR promoter/enhancer, the cytomegalovirus(CMV) immediate early promoter/enhancer [see, e.g., Boshart et al, Cell,41:521-530 (1985)], the SV40 promoter, the dihydrofolate reductasepromoter, the cytoplasmic β-actin promoter and the phosphoglycerolkinase (PGK) promoter.

In another embodiment, inducible promoters may be desired. Induciblepromoters are those which are regulated by exogenously suppliedcompounds, including without limitation, the zinc-inducible sheepmetallothionine (MT) promoter; the dexamethasone (Dex)-inducible mousemammary tumor virus (MMTV) promoter, the T7 polymerase promoter system[WO 98/10088]; the ecdysone insect promoter [No et al, Proc. Natl. Acad.Sci. USA, 93:3346-3351 (1996)]; the tetracycline-repressible system[Gossen et al, Proc. Natl. Acad. Sci. USA, 89:5547-5551 (1992)]; thetetracycline-inducible system [Gossen et al., Science, 268:1766-1769(1995); see also Harvey et al., Curr. Opin. Chem. Biol., 2:512-518(1998)]; the RU486-inducible system [Wang et al., Nat. Biotech.,15:239-243 (1997) and Wang et al., Gene Ther., 4:432-441 (1997)]; andthe rapamycin-inducible system [Magari et al. J. Clin. Invest.,100:2865-2872 (1997)]. Other types of inducible promoters which may beuseful in this context are those which are regulated by a specificphysiological state, e.g., temperature, acute phase, or in replicatingcells only. In a preferred embodiment, the transgene is under thecontrol of the P5 native AAV promoter.

In another embodiment, the native promoter for the transgene will beused. The native promoter may be preferred when it is desired thatexpression of the transgene should mimic the native expression. Thenative promoter may be used when expression of the transgene must beregulated temporally or developmentally, or in a tissue-specific manner,or in response to specific transcriptional stimuli. In a furtherembodiment, other native expression control elements, such as enhancerelements, polyadenylation sites or Kozak consensus sequences may also beused to mimic the native expression.

Another embodiment of the transgene includes transgene operably linkedto a tissue-specific promoter. For instance, if expression in skeletalmuscle is desired, a promoter active in muscle should be used. Theseinclude the promoters from genes encoding skeletal α-actin, myosin lightchain 2A, dystrophin, muscle creatine kinase, as well as syntheticmuscle promoters with activities higher than naturally-occurringpromoters [see Li et al., Nat. Biotech., 17:241-245 (1999)]. Examples ofpromoters that are tissue-specific are known for liver [albumin,Miyatake et al. J. Virol., 71:5124-32 (1997); hepatitis B virus corepromoter, Sandig et al., Gene Ther., 3:1002-9 (1996); alpha-fetoprotein(AFP), Arbuthnot et al., Hum. Gene Ther., 7:1503-14 (1996)], bone[osteocalcin, Stein et al., Mol. Biol. Rep., 2:185-96 (1997); bonesialoprotein, Chen et al., J. Bone Miner. Res., 1:654-64 (1996)],lymphocytes [CD2, Hansal et al., J. Immunol., 161:1063-8 (1998);immunoglobulin heavy chain; T cell receptor α chain], neuronal[neuron-specific enolase (NSE) promoter, Andersen et al. Cell. Mol.Neurobiol., 13:503-15 (1993); neurofilament light-chain gene, Piccioliet al., Proc. Natl. Acad. Sci. USA, 88:5611-5 (1991); theneuron-specific vgf gene, Piccioli et al., Neuron, 15:373-84 (1995)];among others.

Of course, not all vectors and expression control sequences willfunction equally well to express all of the transgenes of thisinvention. However, one of skill in the art may make a selection amongthese expression control sequences without departing from the scope ofthis invention. Suitable promoter/enhancer sequences may be selected byone of skill in the art using the guidance provided by this application.Such selection is a routine matter and is not a limitation of themolecule or construct. For instance, one may select one or moreexpression control sequences, operably link the sequence to a transgeneof interest, and insert the “minigene” comprising the expression controlsequence and the transgene into an AAV vector. After following one ofthe methods for packaging the rAAV taught in this specification, or astaught in the art, one may infect suitable cells in vitro or in vivo.The number of copies of the transgene in the cell may be monitored bySouthern blotting or quantitative PCR; the level of RNA expression maybe monitored by Northern blotting or quantitative RT-PCR; and the levelof protein expression may be monitored by Western blotting,immunohistochemistry, ELISA, RIA, or tests of the transgene's geneproduct's biological activity. Thus, one may easily assay whether aparticular expression control sequence is suitable for a specifictransgene, and choose the expression control sequence most appropriatefor expression of the desired transgene.

C. Production of rAd/AAV Hybrid Virus

The rAd/AAV hybrid virus of the invention may be constructed andproduced using the materials and methods described herein, as well asthose known to those of skill in the art. Such engineering methods usedto construct any embodiment of this invention are known to those withskill in nucleic acid manipulation and include genetic engineering,recombinant engineering, and synthetic techniques. See, e.g., Sambrooket al, and Ausubel et al., cited above; and International PatentApplication No. WO95/13598. Further, methods suitable for producing arAAV cassette in an adenoviral capsid have been described in U.S. Pat.Nos. 5,856,152 and 5,871,982.

Because the rAd/AAV hybrid virus of the invention isreplication-competent, it may be produced by infection and replicationin a selected host cell, using techniques known to those of skill in theart and as described herein. See, U.S. Pat. Nos. 5,856,152 and5,871,982, and discussion of methods of infecting and culturing a hostcell with the rAd/AAV described in Part II below.

II. Production of rAAV

The method of the invention provides for production of rAAV utilizing areplication-competent rAd/AAV. In a particularly preferred embodiment,the rAd/AAV used in the method of the invention provides the rAAVcassette and all necessary adenoviral sequences to the host cell, thusavoiding the need for a second infection or transfection with a helpervirus. Most suitably, the rAd/AAV hybrid virus is engineered asdescribed herein so that all adenoviral gene products required forreplication of the hybrid virus are expressed by the rAd/AAV hybridvirus.

Briefly, the selected host cell is infected with rAd/AAV hybrid virus.Once the rAd/AAV hybrid virus is taken up by a cell, the AAV ITR flankedtransgene must be rescued from the adenovirus backbone by supplying theinfected cell with an AAV rep gene, which preferably is present in thehost packaging cell. The recombinant AAV genome is packaged by supplyingthe infected cell with an AAV cap gene, which is preferably present inthe host packaging cell.

Regardless of the rAd/AAV used in the production of rAAV, critical tothe optimal production of rAAV, the method of the invention includes astep which controls (i.e., inhibits or extinguishes) the ability of therAd/AAV to replicate at a selected time following infection of the hostcells. This step enhances the ability of the rAAV construct carried bythe rAd/AAV to be rescued and packaged into a rAAV virion. This may beachieved by a variety of means, which are described in more detailherein.

A. AAV Rep and Cap Sequences

In order to package the rAAV construct provided by the rAd/AAV hybridinto a rAAV virion, a host cell must contain sequences necessary toexpress AAV rep and AAV cap or functional fragments thereof For example,the rep78/52 proteins may be sufficient to provide the necessary repfunctions. The AAV rep and cap sequences are obtained from an AAV sourceas identified above. The AAV rep and cap sequences may be introducedinto the host cell in any manner known to one in the art as describedabove, including, without limitation, transfection, electroporation,liposome delivery, membrane fusion techniques, high velocity DNA-coatedpellets, viral infection and protoplast fusion. In one embodiment, therep and cap sequences may be transfected into the host cell by one ormore nucleic acid molecules and exist stably in the cell as an episome.In another embodiment, the rep and cap sequences are stably integratedinto the genome of the cell. A stable host cell line that contains repand cap is B-50, described in PCT/US98/19463. Another embodiment has therep and cap sequences transiently expressed in the host cell. Forexample, a useful nucleic acid molecule for such transfection comprises,from 5′ to 3′, a promoter, an optional spacer interposed between thepromoter and the start site of the rep gene sequence, an AAV rep genesequence, and an AAV cap gene sequence.

The rep and cap sequences, along with their expression controlsequences, may be supplied on a single vector, or each sequence may besupplied on its own vector. Preferably, the rep and cap sequences aresupplied on the same vector. Alternatively, the rep and cap sequencesmay be supplied on a vector that contains other DNA sequences that areto be introduced into the host cells.

Preferably, the promoter used in this construct may be any of theconstitutive, inducible or native promoters known to one of skill in theart or as discussed above. In a preferred embodiment, an AAV P5 promotersequence is employed. While it may be obtained from any of theabove-mentioned AAV sources, the parvovirus P5 promoter is preferablyhomologous to the AAV serotype which provides the rep and cap genesequences. Alternatively, the promoter may be a P5 promoter from anotherAAV type than that which provides the rep and cap sequences. AAVs knownto infect other humans or other animals may also provide the P5promoter. The selection of the AAV to provide any of these sequencesdoes not limit the invention.

In another preferred embodiment, the promoter for rep is an induciblepromoter. As discussed above, inducible promoters include, withoutlimitation, the metallothionine (MT) promoter; the dexamethasone(Dex)-inducible mouse mammary tumor virus (MMTV) promoter; the T7polymerase promoter system; the ecdysone insect promoter; thetetracycline-repressible system; the tetracycline-inducible system; theRU486-inducible system; and the rapamycin-inducible system. Onepreferred promoter for rep expression is the T7 promoter. The vectorcomprising the rep gene regulated by the T7 promoter and the cap gene,is transfected or transformed into a cell which either constitutively orinducibly expresses the T7 polymerase. See WO 98/10088, published Mar.12, 1998.

The spacer is an optional element in the design of the vector. Thespacer is a DNA sequence interposed between the promoter and the repgene ATG start site. The spacer may have any desired design; that is, itmay be a random sequence of nucleotides, or alternatively, it may encodea gene product, such as a marker gene. The spacer may contain geneswhich typically incorporate start/stop and polyA sites. The spacer maybe a non-coding DNA sequence from a prokaryote or eukaryote, arepetitive non-coding sequence, a coding sequence withouttranscriptional controls or coding sequences with transcriptionalcontrols. Two exemplary sources of spacer sequences are the λ phageladder sequences or yeast ladder sequences, which are availablecommercially, e.g., from Gibco or Invitrogen, among others. The spacermay be of any size sufficient to reduce expression of the rep78 andrep68 gene products, leaving the rep52, rep40 and cap gene productsexpressed at normal levels. The length of the spacer may therefore rangefrom about 10 bp to about 10.0 kbp, preferably in the range of about 100bp to about 8.0 kbp. To reduce the possibility of recombination, thespacer is preferably less than 2 kbp in length; however, the inventionis not so limited.

Exemplary molecules providing the AAV rep and cap proteins are plasmids,e.g., pMT-Rep/Cap, pP5-Rep/Cap and pMMTV-Rep/Cap. These plasmids eachcontain a neomycin selective marker gene and express the AAV rep/capgenes driven by either their native P5 promoter (pP5-Rep/Cap), thezinc-inducible sheep metallothionine promoter (pMTRep/Cap), or thedexamethasone (Dex)-inducible mouse mammary tumor virus (MMTV) promoter(pMMTV-Rep/Cap). Although these proteins may be provided to the cell byvarious means, exemplary methods of the invention include use of variousplasmids. For construction of plasmid pMT-Rep/Cap, the ORF6 sequence wasremoved from a pMTE4ORF6 plasmid [G. P. Gao et al, J. Virol.,70:8934-8943 (1996)] by BamHI digestion and replaced with a 4.1 kbrep/cap fragment which was prepared by PCR amplification using pSub201plasmid [Samulski, R. J. et al., J. Virol., 63:3822-3828 (1989)] as atemplate. Plasmid pMMTV-Rep/Cap was constructed in the same way aspMT-Rep/Cap, except that a pMMTVE4ORF6 plasmid [Gao et al, cited above]was used as the vector backbone. For construction of P5-Rep/Cap, the MTpromoter and ORF6 sequences were removed from a pMTE4ORF6 plasmid [G. P.Gao et al, J. Virol., 70:8934-8943 (1996)] by EcoRI/BamHI digestion andreplaced with a 4.3 kb P5-Rep/Cap fragment which was isolated from apSub201 plasmid [Samulski, R. J. Et al, J. Virol., 63:3822-3828 (1989)]by XbaI digestion. Plasmid construction involved conventional geneticengineering methods, such as those described in Sambrook et al, citedabove. All of the above-cited references are incorporated by referenceherein.

A variety of other plasmid constructs providing the rep and cap proteinsare known in the art and may be employed in the host cell of theinvention. For example, the rep/cap constructs may omit the spacerbetween the promoter and the rep/cap genes referred to in the constructdescribed above. Other constructs of the art, such as that described inU.S. Pat. No. 5,622,856, which places the P5 promoter 3′ to the rep/capgenes, may also be employed in this context.

The molecule providing the rep and cap proteins may be in any form whichtransfers these components to the host cell. As exemplified herein, thismolecule is preferably in the form of a plasmid, which may contain othernon-viral sequences, such as those for marker genes. This molecule doesnot contain the AAV ITRs and generally does not contain the AAVpackaging sequences. To avoid the occurrence of homologousrecombination, other virus sequences, particularly those of adenovirus,are avoided in this plasmid. This plasmid is desirably constructed sothat it may be stably transfected into a cell.

Although the molecule providing rep and cap may be transientlytransfected into the host cell, it is preferred that the host cell bestably transformed with sequences necessary to express functionalrep/cap proteins in the host cell, e.g., as an episome or by integrationinto the chromosome of the host cell. Depending upon the promotercontrolling expression of such stably transfected host cell, the rep/capproteins may be transiently expressed (e.g., through use of an induciblepromoter).

The methods employed for constructing embodiments of this invention areconventional genetic engineering or recombinant engineering techniquessuch as those described in the references above. While thisspecification provides illustrative examples of specific constructs,using the information provided herein, one of skill in the art mayselect and design other suitable constructs, using a choice of spacers,P5 promoters, and other elements, including at least one translationalstart and stop signal, and the optional addition of polyadenylationsites.

B. Host Cells

The mammalian host cell itself may be selected from any mammalianspecies, such as human cell types, including, without limitation, cellssuch as A549, WEHI, 3T3, 10T1/2, BHK, MDCK, COS 1, COS 7, BSC 1, BSC 40,BMT 10, VERO, WI38, HeLa, 293 cells (which express functional adenoviralE1), Saos, C2C12, L cells, HT1080, HepG2 and primary fibroblast,hepatocyte and myoblast cells derived from mammals including human,monkey, mouse, rat, rabbit, and hamster. The selection of the mammalianspecies providing the cells is not a limitation of this invention; noris the type of mammalian cell, i.e., fibroblast, hepatocyte, tumor cell,etc. The requirements for the cell used is that it must not carry virusgene which could result in homologous recombination of a contaminatingvirus during the production of rAAV; and it must be capable oftransfection of DNA and expression of the transfected DNA.

In a preferred embodiment, the host cell is one that has rep and capstably transfected in the cell, such as the B50 cell line. Other stablerep/cap expressing cell lines, such as those described in U.S. Pat. No.5,658,785, may also be similarly employed. In another suitableembodiment, the host cell is stably transfected with the sequencesnecessary to express any adenoviral gene products necessary forreplication of rAd/AAV hybrid virus lacking from the hybrid virus.

For example, where the rAd/AAV hybrid virus lacks the adenoviral E4 ORF6sequence, the selected cell line is engineered to be stably transfectedwith the sequences necessary for expression of the E4 ORF6 protein. Insuch an instance, the cell line is preferably one which contains thesequences for expression of rep/cap. Following infection of the cell bythe rAd/AAV, the E1a and E1b functions expressed by the hybrid virusturn on expression of the rep/cap functions by the host cell.Thereafter, E4 ORF6 expression is desirably turned on by supplying theagent necessary to induce the promoter controlling E4 ORF6 function toexpress E4 ORF6 gene product.

As discussed herein, this invention may utilize cells of the followingillustrative embodiments:

(a) a cell stably transfected with the AAV rep and cap genes (orfunctional fragments thereof) and the adenovirus E4 ORF6 gene product;

(b) a cell stably transfected with the AAV rep and cap genes (orfunctional fragments thereof), the adenovirus E2a gene product and theadenovirus E4 ORF6;

(c) a cell stably transfected with the AAV rep and cap genes (orfunctional fragments thereof) carried on an episome or integrated intothe chromosomes of the cell and transiently expresses the adenovirus E2agene products;

(d) a cell stably transfected with at least one of the AAV rep and capgenes and the adenovirus E2a gene products (or functional fragmentsthereof); and

(e) a cell stably transfected with the AAV rep gene, the AAV cap gene,E2a gene (or functional fragments thereof) stably as one or moreepisomes or as integrated DNA, and which transiently expresses thetransgene-containing nucleic acid molecule.

Where the host cell and rAd/AAV hybrid virus do not supply the necessaryrep and/or cap sequences and any required adenoviral sequences, thesesequences may be introduced into the host cell by any suitable method,including, for example, transfection, electroporation, liposomedelivery, membrane fusion techniques, high velocity DNA-coated pellets,viral infection and protoplast fusion.

For example, if neither the host cell line nor the rAd/AAV hybrid virusof the invention expresses the E2a gene product, the adenovirus DNAwhich expresses the E2a gene product may be provided to the host cell inthe form of a nucleic acid sequence which also includes a promoterdirecting the expression of the E2a gene product and other optionalregulatory components. The promoter for E2a may be a constitutive,inducible or native promoter, as discussed above. While the promoter incontrol of the expression of the E2a gene product may be a constitutivepromoter in certain embodiments, in one preferred embodiment, thepromoter be an inducible promoter so as to control the amount and timingof E2a gene product generation (which is toxic to the cell uponover-accumulation [D. Brough et al, Virology, 190:624-634 (1992) and D.Klessig et al, Virus Res., 1:169-188 (1984)]) relative to the productionof the E1 gene products. One preferred embodiment provides that thepromoter directing the production of E2a be a different induciblepromoter from that directing the expression of E1a and E1b, and beinducible by exposure to a different inducing agent than that used forthe E1 inducible promoter.

Introduction of a nucleic acid molecule (a plasmid or virus) into thehost cell and the preparation of a host cell useful in this inventionmay also be accomplished using techniques known to the skilled artisanand as discussed throughout the specification. Techniques forconstruction of nucleic acid molecules include cDNA and genomic cloning,which is well known and is described in Sambrook et al. and Ausubel etal., cited above, use of overlapping oligonucleotide sequences of theadenovirus and AAV genomes, combined with polymerase chain reaction,synthetic methods, and any other suitable methods which provide thedesired nucleotide sequence. In preferred embodiment, standardtransfection techniques are used, e.g., CaPO₄ transfection orelectroporation, and/or infection by hybrid adenovirus/AAV vectors intocell lines such as the human embryonic kidney cell line HEK 293 (a humankidney cell line containing functional adenovirus E1 genes whichprovides trans-acting E1 proteins) and the B50 cell lines (a HeLa cellline containing stably integrated rep and cap genes.

D. Methods for Production of rAAV

As described above, the invention provides a method for producingrecombinant adeno-associated virus (rAAV) in the absence ofcontaminating helper virus or wild-type virus. Suitably, the host cellis infected with the rAd/AAV hybrid virus at a multiplicity of infectionin the range of about 0.5 to about 1000 or therebetween, for example,about 1 to about 500, about 10 to about 250, and about 50 to about 200.

The method of producing rAAV virions involves culturing a mammalian hostcell containing a rAd/AAV hybrid virus as described herein whichcontains a rAAV construct to be packaged into a rAAV virion, an AAV repsequence and an AAV cap sequence under the control of regulatorysequences directing expression thereof. Thereafter, the recombinant AAVvirion which directs expression of the transgene is isolated from thecell or cell culture in the absence of contaminating helper virus orwildtype AAV.

Conventional techniques employed in this method include cloning of therAAV viral genomes, and methods of measuring signal generation, and thelike. No purification step is needed to detect message or signal or toseparate the rAAV from other viruses. Generally, in production,conventional purification techniques such as chloride gradientcentrifugation or column chromatography are used to concentrate the rAAVfrom the cellular proteins in the lysate. For example, the cellstogether with transfection medium are harvested by scrapers andsubjected to three rounds of freezing-thawing in ethanol-dry ice and 37°C. water bath. The cells may be centrifuged, e.g., for 15 minutes at 4°C.

Because the hybrid Ad/AAV virus of the invention isreplication-competent, following infection of the selected host cells,in order to optimize production of rAAV virions, it may be desirable tocontrol the ability of the rAd/AAV hybrid virus to replicate, therebyenhancing the ability of AAV construct to be rescued and packaged into arAAV virion. The ability of the rAd/AAV hybrid virus to replicatefollowing infection may be inhibited by a variety of approaches whichwill be readily apparent to those of skill in the art.

For example, in one particularly suitable embodiment, the host cell isinfected with the rAd/AAV hybrid virus containing a temperaturesensitive mutation in an adenoviral gene necessary for adenoviralreplication and/or packaging (e.g., adenoviral gene E2b) at an MOI ofabout 100 to about 200. Thereafter, the host cell is cultured at atemperature of about 32° C. to about 37° C. and rAAV isolated asdescribed herein. As illustrated in the examples below, this method hasbeen found to provide superior yields of rAAV. One example of a suitablerAd containing a temperature-sensitive mutation is sub100r, which hasbeen described [Schaack, J., et al, J. Virol., 69:4079-4085 (1995)].

Additionally, or alternatively, expression of one or more the adenoviralgenes necessary for replication of the rAd/AAV may be controlled. Forexample, the adenoviral E4 (or E4 ORF6) is expressed under the controlof an inducible promoter, by the rAd/AAV or by the host cell where therAd/AAV hybrid virus does not express this gene product. In anotherexample, the adenoviral E1 and E2a gene products are expressed under thecontrol of at least one inducible promoter. Thus, this method furtherincludes the step of contacting the cultured host cells with at leastone inducing agent, which controls the expression of at least one of therequired adenovirus gene products. Where each required adenovirus geneproduct is under control of a different inducible promoter, the methodfurther entails the steps of adding to the host cell culture a firstinducing agent for the first inducible promoter and a second inducingagent for the second inducible promoter. This embodiment of the methodthus permits controlled expression of the adenoviral gene products,e.g., adenoviral genes E1a, E1b, E2a, and/or E4 ORF6. Further, thisinvention permits expression of the adenoviral gene products (e.g.,E1and E1b) in a desired ratio to the expression of the other adenoviralgene product(s) (e.g., E2a) which is optimal for rAAV production in theparticular host cell under suitable culture conditions for that cell.

The determination of a suitable ratio of E1 gene products to E2a geneproducts and the AAV rep/cap products may be accomplished by one ofskill in the art, taking account of the cell type, the strength ofconstitutive and/or inducible promoters used, the amounts of inducer(s)used, and the order or timing of induction of preferred gene products.The optimal ratio which permits the greatest production of rAAV maydiffer as these factors differ. For example, where the E1a gene iscontrolled by a weak or medium strength constitutive promoter, the E2agene should be controlled by a strong inducible promoter and theinducing agent added early in the culture to obtain a suitable ratio.Where the E1a gene is controlled by an inducible promoter as well as theE2a gene, the two inducing agents may be added in varying amounts and atvarying orders of induction to provide the optimal production system forrAAV. However, such optimization experimentation employed to determinepreferred amounts and orders is well within the skill of the art and ismerely routine in light of the disclosures herein.

In another preferred embodiment of the method, the E1a gene product isexpressed under the control of an inducible promoter and the E1b and E2agenes, as well as any other adenoviral genes (e.g., E4ORF6 and/or VAIRNA) that are present, are expressed under the control of their nativepromoter. As discussed above, the E1a gene product activates the nativepromoters of E1b, E2a and any other adenoviral genes. Any induciblepromoter can be used so long as expresses low basal levels of E1a whenthe cell is uninduced and high levels of E1a when the cell is contactedwith an inducing agent. A number of inducible promoters are known in theart and have been discussed throughout the specification. Specificinducible promoters include, without limitation, the zinc-induciblesheep metallothionine (MT) promoter; the dexamethasone (Dex)-induciblemouse mammary tumor virus (MMTV) promoter; the ecdysone insect promoter;the tetracycline-repressible system; the tetracycline-inducible system;the RU486-inducible system; and the rapamycin-inducible system.

The following examples illustrate several preferred methods of theinvention. These examples are illustrative only and are not intended tolimit the scope of the invention.

EXAMPLE 1

Use of the B-50 Cell Line and Ad/AAV Hybrid Vector for Production of aHelper Independent Cell Line

A recombinant Ad/AAV hybrid vector is constructed using the methodsdescribed in U.S. Pat. No. 5,856,152 except that the E3 gene is deletedand the E1 gene operably linked to and under the control of the RSV orPGK promoter is cloned into the E3 region of the adenovirus genome. TheAd/AAV hybrid vector is packaged as described in U.S. Pat. No.5,856,152.

Briefly described, B-50 is a cell which stably expresses AAV type 2 repand cap genes under the control of the homologous p5 promoter. This cellline is characterized by integration of multiple copies (at least 5copies) of P5-rep-cap gene cassettes in a concatamer form into the hostchromosome. This B-50 cell line was deposited with the American TypeCulture Collection, 10801 University Boulevard, Manassas, Va. 20110-2209on Sep. 18, 1997 under Accession No. CRL-12401 pursuant to therequirements of The Budapest Treaty on the International Recognition ofthe Deposit of Microorganisms for the Purposes of Patent Procedure.

B-50 cells are seeded at a density of 2×10⁵ cells per 60 mm plate for 24hours. Twenty-four hours later, the seeding media (DMEM/10% FBSsupplemented with antibiotics) is replaced with DMEM/2% FBS. The cellsare infected with recombinant Ad/AAV clone containing E1 and the rAAVminigene at an appropriate MOI. This one step infection of B-50 cellsprovides all the helper genes required for rAAV production. Thus, therewill be no need for other helper viruses such as sub100r.

Twenty-four hours to ninety-six hours after infection, the cell lysatesare prepared and the lysate is titered for rAAV production by anyprocess known in the art. If the rAAV is rAAVLacZ, the lysate can betitered as follows. The cells together with transfection medium areharvested by scrapers and subjected to three rounds of freezing-thawingin ethanol-dry ice and 37° C. water bath. The cells are centrifuged at3000 rpm in a table top centrifuge for 15 minutes at 4° C. One tenth ofeach lysate is used to infect 84-31 cells, an E1/E4-double complementingcell line which is transducible by rAAV, for 24 hours. The 84-31 cellsare then histochemically stained with X-Gal. The numbers of blue cellsin each infection are scored and presented on the Y-axis of FIG. 1 asInfectious Units (IU, 1 IU was defined as one blue cell counted) ofrAAVLacZ produced in each transfection.

EXAMPLE 2

Production of rAAV in B50 Cells by Replication Competent Ad-AAV HybridVirus

A rAAVCMVGFP genome in which a Green Fluorescent Protein (GFP)reportergene is driven by CMV promoter and flanked by AAV2 ITRs was cloned intothe E3 region of an Ad5 mutant, sub100r virus. The E2b terminal proteingene of sub100r was disrupted by a 3 bp insertion, rendering atemperature sensitive phenotype. The resulting recombinant Ad-AAV hybridis a genotypically wild type for E1, E2a, E4 and VARNA genes but its E3genes are now replaced with a rAAVCMVGFP genome. Thus this secondgeneration Ad-AAV hybrid possesses all essential helper genes and a rAAVgenome and, theoretically, a single infection of B50 cells with thevirus should lead to rescuing, replicating and packaging of rAAVgenomes.

A. Construction of a Replication-competent rAd-AAV Hybrid Virus

A commercially available plasmid construct pAB27 was purchased fromMicrobix Biosystems (Ontario, Canada). It carries the following segmentsof the Ad5 genome: m.u. 0-1, 10.6-16.1, and 69-100 with a 2.7 kb E3deletion spanning m.u. 78.3 n 85.8. A recombinant AAVCMVGFP genome wasisolated from the prAAVCMVGFP construct as a PVU II fragment and clonedin to Sca I site of pAB27 plasmid. The resulting shuttle plasmid wasdesignated as pABrAAVCMVEGFP. On the other hand, an Ad E2B terminalprotein mutant, sub100r was chosen as viral backbone to build up the newAd-AAV hybrid that is conditionally replication competent at itspermissive 32° C. To introduce the rAAVCMVEGFP into the sub100r genome,sub100r viral DNA was digested with restriction endonuclease Spe I andco-transfected with pABrAAVCMVEGFP into 293 cells by calcium phosphatemethod. The transfected cells were overlaid with top agar and culturedat 32° C. for 14 days. The green viral plaques of sub100r-rAAVCMVGFPwere isolated for further plaque purification and expansion to alarge-scale viral prep. Please see the FIG. I for cloning ofpABrAAVCMVGFP construct and illustration of the homologous recombinationprocess to generate subr100rAAVCMVGFP hybrid virus.

B. Production of rAAV

B50 cells were seeded in 12 well plates at a density of 1×10⁵ cells perwell. Twenty-four hours later, the cells were infected with thesub100rAAVCMVGFP hybrid at 100, 1000, 2000, and 4000 viral particles percell. For each infection, triplicates of 12 well plates were set up forincubation at different temperatures, 32, 37 and 39.5° C. As positivecontrols, B50 cells in 12 wells were infected with wild type Ad5 helperand sub100rAAVCMVGFP at the MOIs described above either simultaneouslyor with a 24 hr interval. At 72, 96 and 120 hr post infection, totalcell lysate of each infection was harvested. After have gone throughthree cycles of freeze/thaw, the lysate was spun at 3000 rpm and 4° C.for 15 min. The resulting supernatant of each sample was collected andstored at −80° C. For quantifying rAAVCMVGFP produced in each infection,a portion of each sample was heated at 56° C. for 1 hr to inactivateinfectious Ad-AAV hybrid sub100-rAAVCMVGFP and put onto 84-31 cells, aE1/E4 complementing cell clone, in serial dilution. Green FluorescentForming Units (GFFU) of each sample were scored under an UV-microscopeat 24-48 hr post infection and computed as GFFU per B50 cell used in theinitial sub100rAAVCWGFP infection. One GFFU here was defined by the fociof GFP transduction by rAAVCMVGFP virus in a limiting-dilutioninfection. Thus GFFUs per cell represent rAAVCMVGFP produced by each B50cell under the corresponding experiment set-up.

C. Optimization of Using Replication-competent rAd-AAV Hybrid for AAVProduction in B50 Cells

A series of experiments were set up to optimize conditions for maximumyield of rAAV produced by this new B50/hybrid system and compare withthe original B50/hybrid system.

The B50/Replication competent hybrid infection system can produce about⅓-⅙ of rAAVCMVGFP produced in the presence of Ad helper virus in theinitial experiment. The data summarized in the Table 1 demonstrated thefeasibility of using the B50/replication competent Ad-AAV hybridinfection system for rAAV production.

TABLE 1 Results of initial prove of concept experiment Experiment Set-UpYield of rAAVCMVGFP per cell 72 hr 96 hr 120 hr Ad5 Wt +sub100rAAVCMVGFP, 24 hr interval at 37° C. (positive control)  100 ptseach per cell 360 292 260 1000 pts each per cell 356 288 200 2000 ptseach per cell 136 124 100 4000 pts each per cell 44 40 24sub100rAAVCMVGFP only at 37° C.  100 pts each per cell 13.2 66 70.4 1000pts each per cell 4.6 10.4 9.3 2000 pts each per cell 0.5 1.1 1.3 4000pts each per cell 0.8 1.5 3.7 sub100rAAVCMVGFP only at 32° C.  100 ptseach per cell 0.44 10 50 1000 pts each per cell 0.6 1A 12.8 2000 ptseach per cell 1.5 2.0 7.2 4000 pts each per cell 1.7 3.2 3.6sub100rAAVCMVGFP only at 39.5° C.  100 pts each per cell 0.1 0.1 0.21000 pts each per cell 2.1 2.4 2.7 2000 pts each per cell 1.6 1.4 0.64000 pts each per cell 1.8 1.8 1.2

The data presented here suggest that, under the experiment conditionstested, the B50/replication competent Ad-AAV hybrid system can producerAAV. But the maximum yield went down 3-6 folds as compared to thepositive where B50 cells were infected with an Ad5Wt helper and thehybrid with a 24 hr interval. The single infection system performed alot better at 32 and 37° C. as compared to 39.5° C.

We hypothesized that, once entered B50 cells, replication competentAd-AAV hybrid faces two possible fates: either to be replicated or to becrippled by Rep proteins for AAV rescuing, replicating and packaging. Aproductive infection for rAAV using this system will be a delicatebalance of both directions. We have previously demonstrated that, forhigh yield AAV production by the B50/Ad helper/E1-deleted Ad-AAV hybrid,there is a critical temporal relationship among Rep/cap gene expressiontriggered by Ad5 E1 proteins, transient amplification of incoming Ad-AAVhybrids, resolving of rAAV genomes from the hybrid backbone and furtherreplication of AAV genomes, Although the permissive temperature forsub100r mutant is 32° C., it is known that such temperature sensitivityis somewhat leaky at 37° C. but quite stringent at 39.5° C. We expectedthat, at 37° C., replication of sub100rAAVCMVGFP would be slowed downbut expression of Rep/cap, AAV rescuing, replication and packaging wouldbe optimal. The data demonstrated that the system was indeed moreproductive at 37° C. as compared to 32° C. But AAV productivity at 39.5°C. was sharply decreased. In this case, there was probably no hybridreplication at all. Additionally, the whole cellular machinery was underhigh temperature stress, resulting in deficiency in rep/cap expression,AAV rescuing, replication and packaging.

1. Defining Optimal Multiplicity of Infection for sub100rAAVCMVGFP

Based on the data from Example 2B, infection of B50 cells at lower MOIsappears to be beneficial to production of AAV. According to the datafrom the initial experiment, it appears that, at productivetemperatures, 32 and 37° C., yield of rAAV drops sharply as the MOI ofsub100rAAVCMVGFP infection increases. There is a dramatic difference inAAV productivity between 100 pts/cell and 1000 pts/cell, suggesting thatthe optimal MOI for maximum productivity lie in that range. To definethe optimal MOI, the second experiment carried out was to examine impactof MOI between 20 and 1.000 particles (pt)/cell at both 32 and 37° C.(Table 2). Accordingly, B50 cells were seeded in 12 well plates asdescribed above and infected with 20, 40, 100, 200, 400, and 1000particles of sub100rAAVGFP. The crude cell lysate samples were preparedand assayed for rAAVCMVGFP productivity per cell in the same way asabove.

TABLE 2 Optimization for MOI Experiment Set-up rAAVCMVGFP Yield(GFFU/cell) 72 hr 96 hr 120 hr 144 hr sub100rAAVCMVGFP at 32° C.  20pts/cell 0 0.4 22.1 26.0  40 pts/cell 0 0.4 17.2 30.7  100 pts/cell 0.12.3 40.7 86.7  200 pts/cell 0.1 7.8 26.5 80.6  400 pts/cell 0 9.9 21.422.7 1000 pts/cell 0.2 1.7 4.6 8.2 sub100rAAVCMVGFP at 37° C.  20pts/cell 0.6 2.9 2.5 5.0  40 pts/cell 0.7 8.8 14.5 16.4  100 pts/cell4.2 20.6 27.7 34.7  200 pts/cell 10.1 39.5 62.0 111.3  400 pts/cell 6.721.0 27.1 36.3 1000 pts/cell 2.1 9.0 4.2 12.8 AdsWt + sub100rAAVCMVGFP,a 24 hr interval, at 37° C. (positive control)  100 pts/cell 46.3 100.8119.7 134.4

The results revealed that either 100 or 200 pts/cells was optimal forthe infection at 32° C. But the situation at 37° C. was quite differentwhere the optimal MOI was restrictively limited at 200 pts/cell. Twofolds of increase or decrease of the MOI resulted in a 60% reduction inAAV productivity. The impact of MOI on AAV production observed here andin previous study demonstrated the importance of reaching a delicatebalance between hybrid replication and rAAV packaging in usingB50/hybrid system for AAV production. It is well known that adenovirusreplication process is highly MOI dependent, particularly underpermissive and semi-permissive conditions. It is plausible that, at highMOIs, hybrid virus replication become dominant but rAAV rescuing,replicating and packaging diminish significantly. On the other hand,limited hybrid virus amplification is desirable for increasing thenumber of rAAV genomes for rescuing and packaging. This is clearlydemonstrated in the results presented in Table 2. Since 32° C. ispermissive to sub100rAAVCMVGFP, there was no obvious difference in AAVyield observed between 100 and 200 pts/cell. Only when MOI exceeds 400pts/cell, then AAV yield went down dramatically. However, wheninfections were carried out at a semi-permissive temperature 37° C.,optimal MOI was restricted at 200 pts/cell only. Here, a limited and MOIdependent hybrid virus replication became critical to maximum AAVpackaging.

2. Defining Optimal Temperature for the Single Infection with sub100rAAVGFP

From the initial experiment, it was found that there was very little AAVproduced at 39.5° C. but AAV productivity at 32 and 37° C. wascomparable. To investigate potential impact of infection temperatures onAAV productivity, the MOI experiment described above was run induplicates at both 32 and 37° C.

The data presented in Tables I and 2 also indicated that, at optimalMOIs, AAV production was better at 37° C. than 32° C. This could also beexplained by the need for balancing between limited hybrid virusreplication and AAV rescuing, replicating and packaging. Apparently, atthe semi-permissive temperature 37° C., the equilibrium of two differentevents moved towards in favor of AAV packaging, leading to higherproductivity.

3. Switch of the Temperature from 37 to 32° C. During the InfectionProcess

B50 cells in 12 well plates were infected with sub100rAAVCMVGFP at 100particles per cell and 37° C. for 36 and 60 hr. Then the plates weremoved to another incubator set at 32° C. The crude cell lysate wasprepared at 72, 96, 120, 144 and 168 hr post infection for titration ofAAV productivity on 84-31 cells.

The logic behind this experimental design was that sub100rAAVCMVGFPvirus should have limited replication at 37° C. but activation of P5promoter for Rep/cap protein should be optimal. A 24 hr incubation at37° C. would allow Rep/cap expression initiated with limited level ofhybrid virus replication and thus get B50 cells conditioned for AAVrescuing, replicating and packaging. Once the infection was switched to32° C., hybrid virus replication should also slow down somewhat byinhibition effects of Rep proteins accumulated in the cells. The resultsof the temperature switch experiment (Table 3) did not meet theexpectation probably due to the failure to achieve an optimal balancebetween hybrid replication and AAV rescuing and replicating. But thedata did indicate that an earlier switch in temperature gave rise to abetter yield than a later one, suggesting timing of the switch playcrucial role in AAV production.

TABLE 3 Temperature switching and rAAV productivity rAAVCMVGFPYield-(GFFU/cell) Experiment Set-up 72 hr 96 hr 120 hr 144 hr 168 hrSub100rGFP, 100 pts/cell 00.36 0.4 6.3 15.1 50.4 (37° C. for 36 hr thenswitching to 32° C.) Sub100rGFP,100 pts/cell 1.76 4.4 15.1 15.5 16.6(37° C. for 60 hr then switching to 32° C.) Ad5Wt + 46.3 100.8 119.7134.4 88.2 sub100rAAVCMVGFP (100 pts/cell, a 24 hr interval, 37° C.,positive control)

4. Addition of a Second Batch of sub100rAAVCMVGFP to B50 Cells at 24 HrPost the Initial Infection

B50 cells in 12 well plates were infected with sub100rAAVCMVGFP at 100particle per cell and 37° C. for 24 hr. A second batch ofsub100rAAVCMVGFP at 100 particle per cell was added to 37° C. and theother set was moved to a 32° C. incubator. AAV production under eachcondition was examined at 72, 96, 120, 144 and 168 hr post the initialinfection.

In the classical B50/Ad5 helper/E1-deleted Ad-AAV hybrid system, it wasessential to have a 24 hr interval between the first Ad helper virusinfection and the second Ad-AAV hybrid infection. This delicate temporalrelation of two infections allows creation of optimal cellularconditions, such as proper level of Rep proteins to regulate appropriaterate of hybrid virus replication and AAV rescuing, for high yield of AAVproduction. In that case, the difference between Ad helper virus andE1-deleted hybrid infections is the presence and absence of E1 proteins.However, when replication competent Ad-AAV hybrid is used for AAVproduction in B50 cells, there is no difference between Ad helper virusand the hybrid in terms of their ability to provide all necessary helperfunctions. If we infect the cells with two batches of the hybrid with a24 hr of interval, the first batch should serve as a helper just like AdWt infection and the second batch would be just like E1-deleted hybridto deliver rAAV genomes for amplification, rescuing and packaging. Wewould expect AAV productivity of 850/replication competent Ad-AAV hybridmethod to be as high as that of classical B50/hybrid system. The datagenerated from the experiment was indeed supportive to OUT theory (Table4). Since only one type of Adenovirus used in this new productionmethod, it simplifies our purification process somewhat and eases thescaling-up process. Further more, we could introduce some more severemutations such as E4 deletion into the hybrid backbone and correspondingAd genes into B50 cells stably. Thus we disable the hybrid virus itselfso that even if AAV preps were contaminated with some defective hybrids,side effects of such defective hybrids in vivo would be furtherminimized.

TABLE 4 Improvement of rAAV productivity by a double infection Processwith a single virus, sub100rAAVCMVGFP Experiment Set-up rAAVCMVGFP Yield(GFFU/cell) 72 hr 96 hr 120 hr 144 hr 168 hr Sub100AAVCMVGFP + 0.76 8.7109.2 135.5 88.2 (a 24 hr interval, 100 pts/cell at each infection, 37°C.) Sub100AAVCMVGFP + 9.0 45.2 44.1 54.6 34.7 Sub100AAVCMVGFP (a 24 hrinterval, 100 pts/cell at each infection, 37° C. for 24 hr, switch to32° C. after 2^(nd) infection) Ad5Wt + 46.3 100.8 119.7 134.4 88.2sub100rAAVCMVGFP (100 pts/cell, a 24 by interval, 37° C., positivecontrol)

All documents cited above are herein incorporated by reference. Numerousmodifications and variations of the present invention are included inthe above-identified specification and are expected to be obvious to oneof skill in the art. Such modifications and alterations to the processesof the present invention are believed to be encompassed in the scope ofthe claims appended hereto.

What is claimed is:
 1. A method for producing recombinantadeno-associated virus (rAAV) comprising the step of: culturing a hostcell comprising: (a) an AAV rep sequence and an AAV cap sequence underthe control of regulatory sequences directing expression thereof; and(b) an adenovirus/AAV hybrid virus comprising a recombinantadeno-associated virus (rAAV) vector and nucleic acid sequences encodingadenovirus E1a and adenovirus E1b under the control of regulatorysequences directing expression of the E1a and E1b gene products, whereinsaid hybrid virus contains sufficient adenoviral sequences to permitreplication of said hybrid virus in a host cell.
 2. The method accordingto claim 1, further comprising the step of isolating the rAAV from saidhost cell or host cell culture.
 3. The method according to claim 1,wherein the host cell is stably transformed with the AAV rep sequence.4. The method according to claim 1, wherein the host cell is stablytransformed with the AAV cap sequence.
 5. The method according to claim1, wherein the rep sequence is transiently expressed in the host cell.6. The method according to claim 1, wherein the cap sequence istransiently expressed in the host cell.
 7. A method according to claim1, further comprising the step of, prior to culturing, infecting thehost cell with the hybrid adenovirus/AAV at a multiplicity of infectionof about 0.5 to about
 1000. 8. The method according to claim 1, whereinthe hybrid adenovirus/AAV further comprises a temperature-sensitivemutation in the adenovirus E2b gene.
 9. The method according to claim 1,wherein the hybrid adenovirus/AAV is cultured at a temperature of about32° C. to about 37° C.
 10. The method according to claim 1, wherein theadenovirus/AAV hybrid virus further comprises a functional deletion inthe E3 region.
 11. The method according to claim 1, wherein theadenovirus/AAV hybrid virus further comprises a non-functional deletionof adenoviral coding sequences in the E4 region, wherein said hybridvirus contains the E4 ORF6 region.
 12. The method according to claim 1,wherein the E1a and E1b nucleic acid sequences are located in the siteof the wild-type E3 region of the adenovirus/AAV hybrid virus.
 13. Themethod according to claim 1, wherein the rAAV vector is located in thewild-type adenoviral E1a and E1b region of the adenovirus/AAV hybridvirus.
 14. The method according to claim 1, wherein the AAV vector inthe adenovirus/AAV hybrid virus comprises AAV 5′ and 3′ invertedterminal repeats (ITRs) and a transgene under the control of regulatorysequences directing expression thereof.
 15. The method according toclaim 1, wherein the regulatory sequences in the adenovirus/AAV hybridvirus comprise a first promoter which directs the expression of the E1agene product.
 16. The method according to claim 15, wherein the firstpromoter in the adenovirus/AAV hybrid virus is selected from the groupconsisting of a native promoter of E1a, an inducible promoter, atissue-specific promoter, and a constitutive promoter.
 17. The methodaccording to claim 15, wherein the regulatory sequences in theadenovirus/AAV hybrid virus comprise a second promoter which directs theexpression of the adenoviral E1b gene product.
 18. The method accordingto claim 17, wherein the second promoter in the adenovirus/AAV hybridvirus is identical to the first promoter.
 19. The method according toclaim 17, wherein the second promoter and the first promoter in theadenovirus/AAV hybrid virus are different.
 20. A method for producingrecombinant adeno-associated virus (rAAV), comprising the step of:culturing a host cell containing: (a) an AAV rep sequence and an AAV capsequence under the control of regulatory sequences directing expressionthereof; and (b) an adenovirus/AAV hybrid virus comprising a recombinantadeno-associated viral (rAAV) vector and nucleic acid sequences encodingadenovirus E1a and adenovirus E1b are under the control of regulatorysequences directing expression of the E1a and E1b gene products, whereinsaid hybrid virus contains sufficient adenoviral sequences to permitreplication of said hybrid virus in a host cell; wherein said host cellis cultured under conditions which inhibit hybrid virus replication,resulting in enhanced rAAV production.
 21. The method according to claim20, wherein the adenovirus/AAV virus further comprises a temperaturesensitive mutation in the adenoviral E2b gene.
 22. A method forproducing recombinant adeno-associated virus (rAAV) comprising the stepof: culturing a host cell comprising: (a) an AAV rep sequence and an AAVcap sequence under the control of regulatory sequences directingexpression thereof; and (b) an adenovirus/AAV hybrid virus comprising:(i) adenovirus 5′ cis-elements necessary for replication and packaging;(ii) a deletion of adenoviral sequences in the native adenoviral E1a andE1b region; (iii) a recombinant adeno-associated viral (rAAV) vector;(iv) a deletion of adenoviral sequences from the E3 region; (v) nucleicacid sequences encoding adenovirus E1a and adenovirus E1b under thecontrol of regulatory sequences directing expression of the E1a and E1bgene products, wherein said E1a and E1b nucleic acid sequences arelocated in the site of the E3 region; and (vi) adenovirus 3′cis-elements necessary for replication and packaging.